The complete assembly of human LAT1-4F2hc complex provides insights into its regulation, function and localisation

The LAT1-4F2hc complex (SLC7A5-SLC3A2) facilitates uptake of essential amino acids, hormones and drugs. Its dysfunction is associated with many cancers and immune/neurological disorders. Here, we apply native mass spectrometry (MS)-based approaches to provide evidence of super-dimer formation (LAT1-4F2hc)2. When combined with lipidomics, and site-directed mutagenesis, we discover four endogenous phosphatidylethanolamine (PE) molecules at the interface and C-terminus of both LAT1 subunits. We find that interfacial PE binding is regulated by 4F2hc-R183 and is critical for regulation of palmitoylation on neighbouring LAT1-C187. Combining native MS with mass photometry (MP), we reveal that super-dimerization is sensitive to pH, and modulated by complex N-glycans on the 4F2hc subunit. We further validate the dynamic assemblies of LAT1-4F2hc on plasma membrane and in the lysosome. Together our results link PTM and lipid binding with regulation and localisation of the LAT1-4F2hc super-dimer.

4 Supplementary Figure 2. Probing the glycoforms of LAT1-4F2hc.A) Simulation of LAT1-4F2hc proteoforms.The N-glycan structures with different numbers of Fuc, Neu5Ac and Gal-GlcNAc units were extracted from a previous bottom-up MS analysis of LAT1-4F2hc 10 , and used for simulation of LAT1-4F2hc proteoforms with four N-glycans at the intact protein level.The molecular weight of each proteoform is calculated based on the mass of protein backbone and combination of the four N-glycans.
The simulated proteoforms are firstly sorted in ascending order, and then binned with a window of 5 Da.The mass difference between each proteoform (ΔM) is calculated and plotted as a scatter graph in panel B. The average mass difference between each proteoform is calculated as 72.95 ± 0.69 Da.C) The charge states of +28, +27, and +25 in the spectra of heterodimeric LAT1-4F2hc.The Δm/z between the adjacent peaks is calculated and plotted (scatter plots right-hand side).The results show that each peak differs by ~73 Da for each charge state.Based on these simulations this assessment suggests that these peaks are resolved glycoforms with different monosaccharide compositions.6 Supplementary Figure 3. Deconvolution of LAT1-4F3hc complex using UniDec (panel A, B and C) and iFAMs (panel D,E and F) software.A) The processed mass spectrum with background subtraction (line subtraction: 1).This spectrum is used for UniDec deconvolution.B) The zero-charged spectrum (deconvolved) of LAT1-4F2hc complex with Mass Distribution Smoothing function (mass differences: 72.95; mass smooth width: 10).The major peaks are ~140 kDa.The UniScore (average peaks score) is 56.35 with R 2 of 0.99989.C) The deconvolved spectrum showing LAT1-4F2hc peaks ranging from 136 to 150 kDa.The inset spectrum shows the average mass difference (ΔM ̅̅̅̅ ) is 72.9 Da.D) The unprocessed native mass spectrum (black peaks) overlayed with the iFAMs-reconstructed LAT1-4F2hc peaks with charge +23 to +28 (colored peaks).E) The corresponding FT spectrum shows the charge-state-specific peaks (highlighted with colored boxes).F) The deconvolved zero-charged spectrum.The inset spectrum shows the average mass difference (ΔM ̅̅̅̅ ) is 72.9 Da. experimental LAT1-4F2hc super-dimer spectra, the resolution (m/Δm) of the simulated LAT1-4F2hc super-dimer spectrum was reduced to 2900.This resolution represents the typical resolving power (R=17500 at m/z 200) of a Q-Exactive UHMR mass spectrometer for analyzing a 280 kD membrane protein.(D) The theoretical native mass spectrum of LAT1-4F2hc super-dimer was then simulated at R=17500, (E) The experimental spectrum was compared with the simulated spectrum.(F) There is no significant difference between the theoretical (280954 Da) and experimental molecular weights (280934±109 Da) of LAT1-4F2hc super-dimers.The higher baseline of the experimental mass spectrum of the LAT1-4F2hc super-dimer implies that the super-dimer may still carry multiple detergents/solvent ions.N424 (right panel).The N-glycan compositions are labelled on the corresponding peaks.All N-glycans are core-fucosylated, in line with the previous glycomics and glycoproteomics analysis of the recombinant 4F2hc subunit 10 .B) MS/MS spectrum of a phosphopeptide encompassing 4F2hc-S165.C) MS/MS spectrum of a phosphopeptide containing LAT1-S35.The peaks with neutral loss of 98 Da (H3PO4) are labelled with asterisks.D) Native MS spectrum of the desialylated LAT1-4F2hc complex (5061-5073 m/z, charge state +27).The theoretical peaks of P4 proteoforms (apo, phosphorylated and with additional GlcNAc) and P3 proteoforms (tri-and tetra-fucosylated) were simulated and plotted.
The simulation show that the tri-and tetra-fucosylated P3 proteoforms overlap with the P4 proteoforms with phosphorylation and an additional GlcNAc, respectively.Supplementary Figure 6.Analysis of N-glycan branching and sialylation on LAT1-4F2hc.A) Annotation of the N-glycan branching events on the desialylated LAT1-4F2hc heterodimer (charge state +27).The major peaks differing by a GlcNAc1Gal1 unit are labelled with P1 to P10.Two Gaussian curves can be fitted to the P1 to P10 proteoforms, highlighted with dashed red lines.B) N-glycan branching (GlcNA1Gal1), fucosylation (Fuc1) and sialylation (Neu5Ac1) are the three main features of the N-glycan microheterogeneity in the LAT1-4F2hc complex.C) Calculating the modulus of N-glycan branching, fucosylation and sialylation.D) The correlation between the masses of sialylated LAT1-4F2hc and their modulus (mass mod 73.06).The trend line (linear regression) is shown (red line) with 95% confidence interval (red shaded area).From the modulus analysis, we found that the largest LAT1-4F2hc proteoform carries seven more Neu5Ac residues than the smallest proteoform.E) Comparison of sialylated and desialylated LAT1-4F2hc complexes.Neuraminidase treatment results in a loss of ~3795 Da (~13 Neu5Ac residues) from LAT1-4F2hc complex.These data suggest that the fully glycosylated LAT1-4F2hc complexes carry 9 to 16 Neu5Ac residues on its four N-linked glycans.energy (Source Frag.), In-source trapping energy (IST) and HCD energy (HCD) are labelled.B) Expanded view of the native mass spectra of gas-phase dissociated LAT1 (top panel) and TCEPreduced LAT1 (bottom panel).The higher collision energy used to dissociate LAT1 from the LAT1-4F2hc heterodimer also removes bound phospholipids.C) Mass photometry analysis of glyco-diosgenin (GDN) micelles.GDN micelles with different volumes as shown were diluted into phosphate-buffered saline (PBS) to give a total volume of 20 µl.In both measurements the GDN micelles are < 100 kDa in line with a previous report 11 .This result suggests that empty GDN micelles after dilution do not affect mass photometry analysis for large membrane protein complexes (> 100kDa).D) Native mass spectra of TCEP-reduced LAT1-4F2hc heterodimer under different activation energies.We observed unresolved proteolipomicells at lower activation energy (source fragmentation 50 V), and well-resolved LAT1 and 4F2hc subunits under higher activation energy (source fragmentation 150 V).Supplementary Figure 9. A) Structures of the diacyl-PE and alkyacyl-PE (PE-P) lipids.B) Schematic workflow of in-solution delipidation.In brief, the LAT1-4F2hc assemblies were incubated with 10×CMC OGNG to release loosely bound lipids.The dissociated lipids were further removed after buffer-exchange using 100 kDa MWCO filters (Amicon, Millipore).The delipidated LAT1-4F2hc protein complexes were then analyzed with MS-based lipidomics.C) Relative abundances of PE and PE-P in LAT1-4F2hc samples before and after delipidation (control and delipidation respectively).Supplementary Figure 10.A) Validation of LAT1 palmitoylation observed in mass spectra as an adduct (shaded pink) after expression of the LAT1-4F2hcR183 mutation.Hydroxylamine was used to break thioester bonds between the palmitoyl group and sulfhydryl group at pH 7.0 12 .Following TCEP reduction mass spectra of LAT1 (charge state +15) were recorded as a function of temperature (25 C or 37 C).The palmitoylated proteoform of LAT1 was found to reduce after hydroxylamine treatment, more effecfively at the higher temperature.B) Prediction of palmitoylation site of LAT1 using GPS-Palm software 1 .Only two Cys residues in human LAT1 (C187 and C458) are predicted to carry S- The buried surface area was calculated using PDBePISA server 13 .F) Structure of Adic dimer with interfacial amino acid residues involved in homodimerization highlighted in red.The buried surface area is higher for Adic than LeuT.G) AlphaFold-Multimer 4 predicted two models for the LAT1 homodimer with ipTM score greater than 0.3.The upper and lower models are LeuT-like and Adic-like LAT1 homodimers.The observation of lipid-free LAT1 homo-dimer supports the proposal that the LAT1 homodimerization is Adic-like with a greater buried surface area removing the requirement for lipid binding 14 .B was 0.1% FA in 80% acetonitrile with 20% H2O.The LTQ mass spectrometer was operated in datadependant acquisition mode with one full MS scan (335 to 2000 m/z at a resolution of 60000) followed by MS/MS scans with the collision-induced dissociation (CID) normalized energy of 35%.The glycopeptide identification was performed manually using Xcalibur (version 4.1).The phosphopeptide identification was performed using Maxquant v2.1.0.
Lipidomics analysis.The LAT1-4F2hc complex (20 µg) was buffer-exchanged into 1M ammonium acetate, pH 7.0 with 2 mM OGNG and incubated with 1 µg trypsin overnight at 37 °C.One biological replicate was performed.The digested peptide/lipid mixture was dried using a SpeedVac vacuum concentrator (Thermo Fisher Scientific) and reconstituted with 70% mobile phase A (acetonitrile/H2O: 60/40, 10 mM ammonium formate and 0.1% formic acid) and 30% mobile phase B (isopropanol/acetonitrile: 90/10, 10 mM ammonium formate and 0.1% formic acid) for the following LC-MS/MS analysis.Lipids were directly loaded onto a C18 column (Acclaim PepMap 100, C18, 75 µm × 15 cm, Thermo Scientific) by a Dionex UltiMate 3000 RSLC Nano system coupled to a LTQ Orbitrap mass spectrometer (Thermo Scientific).The lipids were separated with a gradient from 30% to 99 % mobile phase B. For data-dependent acquisition, full MS scans were acquired on the Orbitrap (m/z 400-2000) with a resolution of 60000.Collision-induced dissociation (CID) fragmentation in the ion trap was performed for the five most intense ions at an automatic gain control target of 30,000 and a normalized collision energy of 38%.Raw data were processed with MZmine v2.53 for phospholipid identification and quantification 2 .Lipid quantification for delipidated LAT1-4F2hc was performed manually using Xcalibur 4.4.The extracted ion chromatogram (XIC) of each lipid was processed with 20 ppm mass tolerance and a 7-point Gaussian smoothing.The area under the curve (AUC) was integrated for lipid quantification.
In silico simulation.The sequences of LAT1 and 4F2hc were submitted to AlphaFold Colab (v1.5.2) 3 for LAT1 homodimer and LAT1-4F2hc super-dimer structure prediction using AlphaFold2-multimer 4,5 .For simulation of glycan conformers, the non-glycosylated protein structures were retrieved from RCSB PDB database and prepared using CHARM-GUI 6 .The simulation of glycan conformers on LAT1-4F2hc was performed using GlycoSHIELD (v 0.1) 7 .ChimeraX 1.2.5 was used for protein structure visualization 8 .Data analysis.Simulation and visualization of the pseudo-spectrum of the LAT1-4F2hc super-dimer was performed using Jupyter Notebook with Python 3 and Seaborn library 9 .Statistical analysis was performed using Prism 8.0 (GraphPad Software Inc., San Diego, CA).

89839).
In vivo cross-linking.HeLa cells were harvested and washed three times with ice-cold DPBS (pH 8.0, Gibco, Thermo Fisher Scientific).Cells were then suspended in PBS (pH 8.0) at 2.5 × 10 7 cells/mL and mixed with freshly prepared BS 3 reagent (Thermo Scientific) at a final concentration of 1 mM at room temperature.After 45 min incubation, the reaction was quenched by adding 500 mM Tris buffer (pH 7.4) to a final concentration of 20 mM.After 15 min incubation, the cells were lysed for Western blotting or affinity-purfication experiments.
Preparing the membrane fraction for affinity-purification and Western blotting.A431, HeLa and HepG2 cells were cultured in DMEM and MEM medium (Gibco, Thermo Fisher Scientific) at 37 °C under 5% CO2, until the cell confluency reached 70%.The cells were then harvested and washed three times with ice-cold DPBS (Gibco, Thermo Fisher Scientific).The cells (packed cell volume of 0.2 mL) were then suspended in 0.6 mL hypotonic buffer (10 mM HEPES, 1 mM EGTA, 25 mM KCl, pH 7.8) and incubated at 4 °C for 20 min.After centrifugation at 600 g for 10 min, the cells were suspended in two times the volume of the packed cell in isotonic buffer (10 mM HEPES, 1 mM EGTA, 25 mM KCl and 250 mM sucrose, pH 7.8) with EDTA-free protease inhibitor cocktail (Roche).Then, the cells were homogenized and centrifuged at 1000 g for 10 min at 4 °C to remove the nuclei and unbroken cells.
The supernatant was collected and centrifuged again at 12000 g for 15 min at 4 °C to remove the mitochondrial fraction.The supernatant was diluted 2-fold with PBS and further centrifuged at 150000 g to pellet the membranes for the following affinity-purification and Western blotting experiments.
Affinity-purification of cross-linked endogenous LAT1-4F2hc complexes.Membranes from HeLa cells were incubated with TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.4 ) with 1% DDM at 4 °C for 2 hours then centrifuged at 12000 g for 10 min to remove unsolubilized particles.The supernatant (solubilized membrane proteins) was collected and diluted with TBS buffer to a final concentration of 0.2 % DDM.Solubilized membrane proteins were then incubated with 50 µL Anti-LAT1 antidbody (#5347, Cell Signaling Technology) at 4 °C overnight; then incubated with 50 µL Protein A agarose beads (#9863, Cell Signaling Technology) at 4 °C for 2 hours.After washing three times with TBS buffer containing 0.1% DDM, the beads were incubated with 20 µL 2 × NuPAGE LDS sample buffer (Pierce) at 95 °C for 5 min and loaded to SDS-PAGE gel for separation.
Western blotting of endogenous LAT1-4F2hc assemblies.The membranes of A431, HeLa and HepG2 cells were incubated with 1× NuPAGE LDS sample buffer (Pierce) for 30 min at room temperature and centrifuged at 12000 g for 5 min to remove undissolved particles.To reduce the disulfide bond in the LAT1-4F2hc complex, the samples in LDS sample buffer were further incubated with 10 mM DTT at 56 °C for 10 min.Membrane proteins were then separated by NuPAGE 4 to 12%, Bis-Tris gel (Invitrogen) and transferred to PVDF membrane (Invitrogen).The PVDF membrane was then blocked with 5 % BSA (Fraction V, Roche) in TBST buffer (50 mM Tris, 150 mM NaCl and 0.1% Tween-20) and incubated with primary antibodies diluted 1000 fold with TBST supplemented with 0.1% BSA overnight at 4 °C.The following antibodies Anti-LAT1 (#5347), Anti-4F2hc antibody palmitoylation.C) Structural illustration of LAT1-C187, LAT1-C58, 4F2hc-R183 and the interfacial PE lipid.LAT1-C187 is in close vicinity of 4F2hc-R183, whereas LAT1-C458 is distal to 4F2hc-183.The interfacial PE is between LAT1-C187 and 4F2hc-R183.Therefore, we propose that the mutation of LAT1-R183L, which abolishes interfacial PE binding, induces palmitoylation on LAT1-C187.18 Supplementary Figure 12. Analysis of possible phospho-regulation of LAT1 dimerisation and interactions with PE.A) Structural illustration of LAT1-4F2hc complex with PE binding and phosphorylation on LAT1-S35.B) The spectrum of non-glycosylated LAT1-4F2hc 4M complex without and with PE binding (charge state +26).The peaks corresponding to phosphorylated proteoforms are labeled (red circles).The phosphorylation levels of apo-and PE-bound forms of LAT1-4F2hc 4M complex are plotted with error bars showing mean ± standard deviation from three independent experiments (dots).C) Native mass spectrum of LAT1 monomers and homo-dimers.D) Comparison of phosphorylation levels of monomeric (charge state +14) and homo-dimeric LAT1 (charge state +21).The theoretical phosphorylation level of the LAT1 homo-dimer was simulated based on the abundance of phosphorylated LAT1 monomer.Bars show mean ± standard deviation from three independent experiments (dots).E) Structure of the LeuT dimer.Interfacial phospholipid binding is critical for LeuT dimerization and the amino acids in the homo-dimer interface are highlighted in red.